Cobalamin (Cbl) or vitamin B.sub.12 deficiency, usually the result of disruption of the absorption of cobalamin, can lead to life-threatening hematological and neuropsychiatric abnormalities. Accurate and early diagnosis of cobalamin deficiency is important since proper treatment with cobalamin results in complete reversal of the hematologic symptoms. Early diagnosis is especially important in order to avoid potentially incapacitating, irreversible neurologic damage. Administration of exogenous cobalamin always stops the progression of neuropsychiatric abnormalities, almost always leads to significant improvements in such symptoms and frequently leads to their complete correction. Early diagnosis is often difficult since the clinical signs of Cbl deficiency can result from a variety of other disorders. It has generally been taught that Cbl deficiency should be suspected in individuals with significant anemia, displayed for example in decreased hematocrit or hemoglobin, with macrocytic red blood cells (i.e., mean cell volume (MCV) generally greater than 100 fl), or in individuals having the neurologic symptoms of peripheral neuropathy and/or ataxia. Anemia associated with Cbl deficiency has been described as typically severe with hemoglobin .ltoreq.8 g % or hematocrit &lt;25% and the size of the red blood cells is described as greatly increased to levels &gt;110 fl. (See, for example, Babior and Bunn (1983) in Harrison's Principles of Internal Medicine (Petersdorf et al., eds.) McGraw-Hill Book Co., New York; Lee and Gardner ( 1984) In Textbook of Family Practice, 3rd Ed. (Rakel, ed.) Saunders & Co., Philadelphia).
Cbl deficiency is often difficult to distinguish from folate deficiency because both lead to indistinguishable hematologic abnormalities. It is very important to make this distinction because treatment with the proper vitamin will result in the greatest improvement in hematologic symptoms and the neuropsychiatric abnormalities associated with Cbl deficiency are only corrected by cobalamin treatment. Furthermore, the incorrect treatment of Cbl deficiency with folate can be dangerous in that folate treatment may improve some, or all, of the hematologic abnormalities thereby disguising the presence of Cbl deficiency and delaying timely treatment with cobalamin. As noted above, delay in cobalamin treatment can in some cases result in irreversible neurologic damage.
The serum cobalamin assay has been essentially the only laboratory assay generally available for use in determining if a patient is Cbl deficient. Presently preferred cobalamin assays are radiodilution assays which use pure or purified intrinsic factor as the binding protein (see: Kolhouse et al. (1978) New Eng. J. Med. 299:785-792). This assay has been criticized as frequently giving low Cbl values in patients who lack any evidence of Cbl deficiency. It has been suggested (Schilling et al. (1983) clin. Chem. 29:582-583) that this assay may frequently give false positives showing low serum Cbl levels in individuals who are not Cbl deficient.
It has long been known that methylmalonic acid (MMA) is excreted in increased amounts in the urine of most patients with Cbl deficiency (see, for example, Cox and White (1962) Lancet ii:853-856; Norman et al. (1982) Blood 59:1128-1131). In Cbl deficiency, reduced levels of adenosyl-Cbl result in decreased activity of L-methylmalonyl-coenzyme A (CoA) mutase and a concomitant increase in intracellular levels of L-methylmalonyl-CoA. D-methylmalonyl-CoA, resulting from transformation of the L-isomer by D,L-methylmalonyl-CoA racemase (Stabler et al. (1985) Arch. Biochem. Biophys. 241:252-264), is cleaved to CoA and MMA by D-methylmalonyl-CoA hydrolase which has been recently characterized (Kovachy et al. (1983) J. Biol. Chem. 228:11415-11421). MMA is then released into blood in unknown amounts and is excreted in the urine. About 70% of the MMA in blood is metabolized to unknown products via as yet undefined pathways and only about 30% is excreted in the urine.
Recently, Marcell et al. (1985) Anal. Biochem. 158:58-66 reported that MMA in the serum and urine of normal subjects ranged from 19-76 ng/ml and 270-7190 ng/ml, respectively. Stabler et al. a. (1986) J. Clin. Invest. 77:1606-1612 reported that serum MMA levels of clinically confirmed Cbl deficient patients ranged from 55 to 22,300 ng/ml, with 69 of 73 of such patients having serum MMA levels above the normal range. It was also reported that there was a positive correlation between serum MMA levels and the presence of neurologic abnormalities in these patients. Lindenbaum et al. (1988) New Eng. J. Med. 318:1720-1728 reported that serum MMA levels were elevated above normal levels in 36 of 37 Cbl deficient patients who displayed neuropsychiatric abnormalities in the absence of anemia or other severe hematologic abnormalities. Further, it was suggested that high serum MMA levels which return to normal after cobalamin therapy provide a useful confirmation of the presence of Cbl deficiency.
Stabler et al. (1989) "Marked Elevation of Methylmalonic Acid in Cerebral Spinal Fluid of Patients with Cobalamin Deficiency" Abstract for the meeting of the American Society for Clinical Investigation (Clinical Research (1989) 37:550A), have very recently determined MMA levels in cerebral spinal fluid (CSF) of normal (non-Cbl deficient) and Cbl deficient patients. It was found that MMA levels in CSF are significantly elevated in patients with confirmed Cbl deficiency. It was also found that in Cbl deficient patients, MMA CSF levels were elevated to a greater degree than were MMA serum levels. Further, in several patients having high serum MMA due to renal failure, MMA levels in CSF were within the normal range or only slightly elevated.
The diagnosis of folate deficiency has, likewise, been almost entirely dependent on the demonstration of low serum levels of the vitamin in patients with supportive clinical and laboratory findings. Thus, the most widely utilized and recommended assays for diagnosing and distinguishing Cbl and folate deficiency have been assays of serum levels of the vitamins. Like Cbl deficiency, the diagnosis of folate deficiency can be problematic. For instance, after acute dietary deprivation, serum folate levels may be decreased, even though tissue folate levels are adequate (Herbert (1962) Arch. Intern. Med. 110:649-652). In the setting of chronic alcoholism, the laboratory features of megaloblastic anemia due to folate deficiency may be confused by concurrent illness. Serum and red blood cell folate levels may be normal in patients with alcoholism and megaloblastic anemia.
The sulfhydryl amino acids are metabolized by a complex set of pathways (see: Stabler et al. (1988) J. clin. Invest. 81:466-474) in some of which cobalamin and folic acid are vital cofactors. Methylation of homocysteine (Hcys) to form methionine which is catalyzed by methionine synthetase requires methylcobalamin (Me-Cbl). The methyl group is donated by N.sup.5 -methyltetrahydrofolate, which is converted to tetrahydrofolate (THF). Thus, both cobalamin and folate are cofactors in sulfhydryl amino acid metabolism and cobalamin, but not folate, is a cofactor in methylmalonyl-CoA metabolism. Elevated serum and urine levels of homocystine and/or methionine have been reported in children having inherited defects due to inability to synthesize the cofactors N.sup.5 -methyltetrahydrofolate or Me-cobalamin or to defects in methionine synthetase (see: Mudd (1974) in Heritable Disorders of Amino Acid Metabolism: Patterns of Clinical Expression and Genetic Variation (Nyhan, ed.) John Wiley & Son, New York).
Stabler et al. (1988) supra reports that elevated levels of serum total homocysteine correlate with the presence of either Cbl deficiency or folate deficiency. Most patients with confirmed Cbl deficiency (77 of 78) or folate deficiency (18 of 19) were found to have total serum Hcys levels above the normal range of 7-22 .mu.mol/liter. This reference also reports that measurement of both serum MMA and total Hcys levels can be used to distinguish between Cbl and folate deficiency, since most patients having only folate deficiency have normal serum MMA levels.
Marcell et al. (1985) supra; Stabler et al. (1987) Anal. Biochem. 162:185-196; and Allen et al. U.S. Pat. application Ser. No. 933,553 describe gas chromatography/mass spectrometry selected ion monitoring (GC/MS-SIM) methods for quantitating serum and urine levels of total homocysteine and methylmalonic acid. The assay methods described, particularly that for MMA, require multistep, time-consuming laborious sample preparation due, for the most part, to the low concentration of MMA and Hcys in serum and to the presence in serum of compounds which interfere with quantitation. Homocysteine analysis in body fluids is further complicated since homocysteine readily forms disulfide bonds with itself, other sulfhydryl amino acids (i.e., cysteine) and free sulfhydryl groups in proteins present in such fluids. Separate, different sample preparations are required for the analysis of the two compounds. Development of improved rapid and reproducible assays for serum MMA and Hcys would be desirable. Improvements that would allow combined assay of MMA and homocysteine and which would be amenable to automation of the assay methods would be particularly desirable.
Elevation of MMA levels in urine and serum can result from renal insufficiency. Similarly, Hcys has been reported to be present in small amounts, compared to undetectable levels in normal serum, in serum of patients with renal insufficiency. Renal insufficiency is known to correlate with the level of creatinine in serum. A number of methods for the assay of creatinine are known and the most widely employed assays are based on colorimetric detection. A determination of the presence of renal insufficiency, for example, by assay of creatinine would be useful additional information in the diagnosis of Cbl and folate deficiency. Adaptation and combination of a quantitative assay for creatinine with GC/MS methods of quantitation of MMA and Hcys would be of significant practical utility.